Determination of Aflatoxin B1-Lysine in Pig Serum and Plasma by Liquid Chromatography–Tandem Mass Spectrometry

Mayra C. Di Gregorio, Alessandra V. Jager, Aline A. Costa, Keliani Bordin1, George E. Rottinhghaus, Tânia Petta, Pollyana C.M.C. Souto, Fabio E. L. Budiño and Carlos A. F. Oliveira


Aflatoxin B1 (AFB1) is a hepatocarcinogen produced by certain Aspergillus species growing on crops. After biotransformation in the liver, AFB1generates several metabolites, one of which is AFB1 bound to lysine on serum albumin. AFB1-lysine (AFB1-lys) is a digest product of AFB1-albumin and is considered a biomarker of exposure to AFB1 in humans and animals. The objectives of this paper were to evaluate the performance characteristics of a new analytical method for determination of AFB1-lys levels in pig serum, heparinized and ethylenediaminetetraacetic acid (EDTA) plasma and to evaluate the interference of these anticoagulants in AFB1-lys quantification. Blank blood samples were obtained from eight crossbreed 91-day-old barrows fed AFB1-free diets. Pooled samples (n = 3) and individual samples of serum, EDTA and heparinized plasma collected from five pigs were enzymatically digested with pronase at 37°C for 4 h. AFB1-lys was isolated by solid-phase extraction and quantified by liquid chromatography coupled to tandem mass spectrometry. The analytical method was applied for determination of AFB1-lys in serum and EDTA plasma collected from five 49-day-old crossbreed barrows fed ad libitum diets containing 1.1 mg of AFB1 per kg of feed during 7 days (three animals) or 42 days (two animals). Samples of heparinized plasma were only available from animals intoxicated for 42 days. All animals had lower levels of AFB1-lys in EDTA plasma samples (24.78–37.40 ng/mL), when compared to serum (49.32–252.07 ng/mL−1) or heparinized plasma (176.81 and 264.24 ng/mL−1). EDTA did not interfere in AFB1-lys standard detection, but our findings suggest that EDTA should be avoided during blood collection since it affects the pronase activity in AFB1–albumin adduct digestion and, consequently, causes a reduction in the AFB1-lys levels. Hence, determination of AFB1-lys in serum and heparinized plasma is an approach to assess an individual's exposure of swine to AFB1.

Fonte: Journal of Analytical Toxicology, p.1-6, 2016.

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